Antidotes to antisense compounds

ABSTRACT

The present invention relates to antisense antidote compounds and uses thereof. Such antidote compounds reduce the magnitude and/or duration of the antisense activity of an antisense compound.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/740,974, filed Sep. 13, 2010, now U.S. Pat. No. 8,389,488, issuedMar. 5, 2013, which is a 35 U.S.C. §371 national phase application ofinternational application serial no. PCT/US2008/082511, filed on Nov. 5,2008, which is a non-provisional of U.S. patent application Ser. No.60/985,595, filed on Nov. 5, 2007, the disclosure of each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0076USC1SEQ.txt, created Mar. 4, 2013, which is 4.0 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides methods and compositions for modulatingantisense activity.

BACKGROUND OF THE INVENTION

Antisense compounds have been used to modulate target nucleic acids.Antisense compounds comprising a variety of modifications and motifshave been reported. In certain instances, such compounds are useful asresearch tools and as therapeutic agents.

SUMMARY OF THE INVENTION

In certain embodiments, provided herein are antidote compounds. Suchcompounds reduce antisense activity of an antisense compound. In certainembodiments, the present invention provides antidote compounds that arecomplementary to antisense compounds.

In certain embodiments, the present invention provides antidotecompounds comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence complementary to anantisense compound.

In certain such embodiments, the modified oligonucleotide is asingle-stranded oligonucleotide and/or is at least 90% complementary tothe antisense compound. In certain embodiments, the antidote compound isfully complementary to the antisense compound.

In certain embodiments, antidote compounds at least one internucleosidelinkage of an antidote compound is a modified internucleoside linkage.In certain such embodiments, at least one internucleoside linkage is aphosphorothioate internucleoside linkage.

In certain embodiments, antidote compounds comprise at least onenucleoside comprising a modified sugar. In certain such embodiments, themodified sugar is a bicyclic sugar or sugar comprising a2′-O-methoxyethyl.

In certain embodiments, antidote compounds comprise at least onenucleoside comprising a modified nucleobase. In certain suchembodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, antidote compounds comprise at least onemodification. In certain such embodiments, antidote compounds compriseone or more nucleoside modifications and or one or more linkagemodifications. In certain embodiments, antidote compounds comprise oneor more modifications selected from: sugar modifications, linkagemodifications and nucleobase modifications.

In certain embodiments, antidote compounds comprise a modifiedoligonucleotide comprising: a gap segment consisting of linkeddeoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a3′ wing segment consisting of linked nucleosides; wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment and wherein each nucleoside of each wing segment comprises amodified sugar.

In certain embodiments, antidote compounds comprise a modifiedoligonucleotide comprising: a gap segment consisting of ten linkeddeoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar; and wherein each internucleosidelinkage is a phosphorothioate linkage.

In certain embodiments, antidote compound of any of the above claims,comprise a modified oligonucleotide consisting of 20 linked nucleosides.

In certain embodiments, antidote compound comprise a modifiedoligonucleotide wherein each nucleoside is modified.

In certain embodiments, antidote compounds are complementary to anantisense compound, wherein the antisense compound is targeted to anmRNA. In certain embodiments, the antisense compound is targeted to anmRNA encoding a blood factor. In certain embodiments, the antisensecompound is targeted to an mRNA encoding a protein involved inmetabolism. In certain embodiments, the antisense compound is targetedto an mRNA encoding a protein involved in diabetes. In certainembodiments, the antisense compound is targeted to an mRNA encoding aprotein involved in cardiopathology. In certain embodiments, theantisense compound is targeted to an mRNA encoding a protein expressedin nerve cells. In certain embodiments, the antisense compound istargeted to an mRNA encoding a protein expressed in the central nervoussystem. In certain embodiments, the antisense compound is targeted to anmRNA expressed in peripheral nerves.

In certain embodiments, the antisense compound is targeted to an mRNAencoding a protein expressed in the liver. In certain embodiments, theantisense compound is targeted to an mRNA encoding a protein expressedin the kidney.

In certain embodiments, the antisense compound is targeted to apre-mRNA. In certain embodiments, the antisense compound is targeted toa micro-RNA. In certain embodiments, the antisense compound is an RNaseH dependent antisense compound. In certain embodiments, the antisensecompound alters splicing of a target nucleic acid. In certainembodiments, the antisense compound activates the RISC pathway.

In certain embodiments, antidote compounds activate RNase H. certainembodiments, antidote compounds activate the RISC pathway.

In certain embodiments, the invention provides a composition comprisingan antidote compound or a salt thereof and a pharmaceutically acceptablecarrier or diluent.

In certain embodiments, the invention provides methods comprisingadministering to an animal an antidote compound or composition. Incertain embodiments, the animal is a human. In certain embodiments, theadministering is oral, topical, or parenteral.

In certain embodiments, the invention provides methods of inhibitingantisense activity in a cell comprising contacting the cell with anantidote compound according the present invention and thereby inhibitingthe antisense activity in the cell. In certain such embodiments, thecell is in an animal. In certain embodiments, the animal is a human.

In certain embodiments, the invention provides methods comprising:contacting a cell with an antisense compound; detecting antisenseactivity; and contacting the cell with an antidote compound. In certainembodiments, the method the detecting antisense activity comprisesmeasuring the amount of target mRNA present, the amount of targetprotein present, and/or the activity of a target protein. In certainembodiments, such methods comprising detecting antidote activity bymeasuring antisense activity after contacting the cell with antidotecompound. In certain such methods, the cell is in an animal. In certainembodiments, the animal is a human.

In certain embodiments, the invention provides methods of ameliorating aside-effect of antisense treatment comprising: contacting a cell with anantisense compound; detecting a side-effect; contacting the cell with anantidote compound; and thereby ameliorating the side effect of theantisense compound.

In certain embodiments, the invention provides methods of treating apatient comprising: administering to the patient an antisense compound;monitoring the patient for antisense activity; and if the antisenseactivity becomes higher than desired, administrating an antidotecompound. In certain such embodiments, the monitoring antisense activitycomprises measuring the amount of target mRNA present, measuring theamount of target protein present and/or measuring the activity of atarget protein. In certain embodiments, such methods include detectingantidote activity by measuring antisense activity after administrationof the antidote compound. In certain embodiments, the patient is ahuman.

In certain embodiments, the invention provides methods of treating apatient comprising: administering to the patient an antisense compound;monitoring the patient for one or more side effect; and if the one ormore side effect reaches an undesirable level, administrating anantidote compound. In certain such embodiments, the patient is a human.

In certain embodiments, the invention provides a kit comprising anantisense compound and an antidote compound; an antidote compound and anon-oligomeric antidote; or an antisense compound, an antidote compound,and a non-oligomeric antidote. In certain such embodiments, thenon-oligomeric antidote is a target protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Fas RNA levels in livers of mice after antisense treatmentwith and without subsequent antidote treatment as discussed in Example3. Results are expressed as percent of control mice.

FIG. 2 shows Kinetics of Fas antisense and antidote activity asdiscussed in Example 4.

FIG. 3 shows Fas mRNA after treatment with antisense or mismatchantisense, followed by antidote or mismatch antidote as discussed inExample 4. The mismatch control oligonucleotide did not demonstrateantidote activity.

FIG. 4 shows PTEN mRNA levels following antisense treatment in micefollowed by antidote treatment or by control injection as described inExample 6.

FIG. 5 shows PTEN mRNA levels in mice following treatments described inExample 6, demonstrating that (1) antisense compound ISIS 116847 reducedPTEN mRNA in mouse liver, (2) antidote compound ISIS 126525 partiallyrestored PTEN mRNA by 3 days, and (3) ISIS 40169, which is used here asa non-sense control did not restore PTEN mRNA.

FIG. 6 shows total prothrombin RNA from livers of mice at 3 daysfollowing antisense and antidote treatment as described in Example 10.Data are expressed as percent of saline treated (antisense control).Zero on the X-axis represents no antidote (antidote control).

FIG. 7 shows total thrombin generation three days after treatment withantidote or with control oligonucleotide as described in Example 11.

FIG. 8 shows results of prothrombin time (PT-INR) calculations describedin Example 12.

FIG. 9 shows results of activated partial thromboplastin time (aPPT)calculations described in Example 12.

FIG. 10 shows the total prothrombin RNA three days after injection ofnon-complementary antidotes as described in Example 10. Resultsdemonstrate the specificity of antidote activity.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

DEFINITIONS

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,18th edition, 1990; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosureherein are incorporated by reference in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, the term “nucleoside” means a glycosylamine comprising anucleobase and a sugar. Nucleosides includes, but are not limited to,naturally occurring nucleosides, abasic nucleosides, modifiednucleosides, and nucleosides having mimetic bases and/or sugar groups.

As used herein, the term “nucleotide” refers to a glycosomine comprisinga nucleobase and a sugar having a phosphate group covalently linked tothe sugar. Nucleotides may be modified with any of a variety ofsubstituents.

As used herein, the term “nucleobase” refers to the base portion of anucleoside or nucleotide. A nucleobase may comprise any atom or group ofatoms capable of hydrogen bonding to a base of another nucleic acid.

As used herein, the term “heterocyclic base moiety” refers to anucleobase comprising a heterocycle.

As used herein, the term “oligomeric compound” refers to a polymericstructure comprising two or more sub-structures and capable ofhybridizing to a region of a nucleic acid molecule. In certainembodiments, oligomeric compounds are oligonucleosides. In certainembodiments, oligomeric compounds are oligonucleotides. In certainembodiments, oligomeric compounds are antisense compounds. In certainembodiments, oligomeric compounds are antidote compounds. In certainembodiments, oligomeric compounds comprise conjugate groups.

As used herein “oligonucleoside” refers to an oligonucleotide in whichthe internucleoside linkages do not contain a phosphorus atom.

As used herein, the term “oligonucleotide” refers to an oligomericcompound comprising a plurality of linked nucleosides. In certainembodiment, one or more nucleotides of an oligonucleotide is modified.In certain embodiments, an oligonucleotide comprises ribonucleic acid(RNA) or deoxyribonucleic acid (DNA). In certain embodiments,oligonucleotides are composed of naturally- and/ornon-naturally-occurring nucleobases, sugars and covalent internucleosidelinkages, and may further include non-nucleic acid conjugates.

As used herein “internucleoside linkage” refers to a covalent linkagebetween adjacent nucleosides.

As used herein “naturally occurring internucleoside linkage” refers to a3′ to 5′ phosphodiester linkage.

As used herein, the term “antisense compound” refers to an oligomericcompound that is at least partially complementary to a target nucleicacid molecule to which it hybridizes. In certain embodiments, anantisense compound modulates (increases or decreases) expression oramount of a target nucleic acid. In certain embodiments, an antisensecompound alters splicing of a target pre-mRNA resulting in a differentsplice variant. Antisense compounds include, but are not limited to,compounds that are oligonucleotides, oligonucleosides, oligonucleotideanalogs, oligonucleotide mimetics, and chimeric combinations of these.Consequently, while all antisense compounds are oligomeric compounds,not all oligomeric compounds are antisense compounds.

As used herein, the term “antisense oligonucleotide” refers to anantisense compound that is an oligonucleotide.

As used herein, the term “antisense activity” refers to any detectableand/or measurable activity attributable to the hybridization of anantisense compound to its target nucleic acid. Such detection and ormeasuring may be direct or indirect. For example, in certainembodiments, antisense activity is assessed by detecting and ormeasuring the amount of target protein. In certain embodiments,antisense activity is assessed by detecting and/or measuring the amountof target nucleic acids and/or cleaved target nucleic acids and/oralternatively spliced target nucleic acids.

As used herein the term “detecting antisense activity” or “measuringantisense activity” means that a test for detecting or measuringantisense activity is performed on a particular sample and compared tothat of a control sample. Such detection and/or measuring may includevalues of zero. Thus, if a test for detection of antisense activityresults in a finding of no antisense activity (antisense activity ofzero), the step of “detecting antisense activity” has nevertheless beenperformed.

As used herein the term “control sample” refers to a sample that has notbeen contacted with a reporter oligomeric compound.

As used herein, the term “motif” refers to the pattern of unmodified andmodified nucleotides in an oligomeric compound.

As used herein, the term “antidote compound” refers to an oligomericcompound that is complementary to and capable of hybridizing with anantisense compound.

As used herein, the term “non-oligomeric antidote” refers to a compoundthat does not hybridize with an antisense compound and that reduces theamount or duration of an antisense activity. In certain embodiments, anon-oligomeric antidote is a target protein.

As used herein, the term “antidote activity” refers to any decrease inintensity or duration of any antisense activity attributable tohybridization of an antidote compound to an antisense compound.

As used herein, the term “chimeric antisense oligomer” refers to anantisense oligomeric compound, having at least one sugar, nucleobase orinternucleoside linkage that is differentially modified as compared toat least on other sugar, nucleobase or internucleoside linkage withinthe same antisense oligomeric compound. The remainder of the sugars,nucleobases and internucleoside linkages can be independently modifiedor unmodified, the same or different.

As used herein, the term “chimeric antisense oligonucleotide” refers toan antisense oligonucleotide, having at least one sugar, nucleobase orinternucleoside linkage that is differentially modified as compared toat least on other sugar, nucleobase or internucleoside linkage withinthe same antisense oligonucleotide. The remainder of the sugars,nucleobases and internucleoside linkages can be independently modifiedor unmodified, the same or different.

As used herein, the term “mixed-backbone oligomeric compound” refers toan oligomeric compound wherein at least one internucleoside linkage ofthe oligomeric compound is different from at least one otherinternucleoside linkage of the oligomeric compound.

As used herein, the term “target protein” refers to a protein, themodulation of which is desired.

As used herein, the term “target gene” refers to a gene encoding atarget protein.

As used herein, the term “target nucleic acid” refers to any nucleicacid molecule the expression or activity of which is capable of beingmodulated by an antisense compound. Target nucleic acids include, butare not limited to, RNA (including, but not limited to pre-mRNA and mRNAor portions thereof) transcribed from DNA encoding a target protein, andalso cDNA derived from such RNA, and miRNA. For example, the targetnucleic acid can be a cellular gene (or mRNA transcribed from the gene)whose expression is associated with a particular disorder or diseasestate, or a nucleic acid molecule from an infectious agent.

As used herein, the term “target antisense compound” refers to anantisense compound that is targeted by an antidote compound.

As used herein, the term “targeting” or “targeted to” refers to theassociation of an antisense compound to a particular target nucleic acidmolecule or a particular region of nucleotides within a target nucleicacid molecule.

As used herein, the term “nucleobase complementarity” refers to anucleobase that is capable of base pairing with another nucleobase. Forexample, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.

As used herein, the term “non-complementary nucleobase” refers to a pairof nucleobases that do not form hydrogen bonds with one another orotherwise support hybridization.

As used herein, the term “complementary” refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through nucleobase complementarity. In certain embodiments,an antisense compound and its target are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%complementary to a target nucleic acid.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid or an antidote to its antisense compound). While not limited to aparticular mechanism, the most common mechanism of pairing involveshydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases (nucleobases). For example, the natural base adenine isnucleobase complementary to the natural nucleobases thymidine and uracilwhich pair through the formation of hydrogen bonds. The natural baseguanine is nucleobase complementary to the natural bases cytosine and5-methyl cytosine. Hybridization can occur under varying circumstances.

As used herein, the term “specifically hybridizes” refers to the abilityof an oligomeric compound to hybridize to one nucleic acid site withgreater affinity than it hybridizes to another nucleic acid site. Incertain embodiments, an antisense oligonucleotide specificallyhybridizes to more than one target site.

As used herein, “designing” or “designed to” refer to the process ofdesigning an oligomeric compound that specifically hybridizes with aselected nucleic acid molecule.

As used herein, the term “modulation” refers to a perturbation offunction or activity when compared to the level of the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing.

As used herein, the term “expression” refers to all the functions andsteps by which a gene's coded information is converted into structurespresent and operating in a cell. Such structures include, but are notlimited to the products of transcription and translation.

As used herein, “variant” refers to an alternative RNA transcript thatcan be produced from the same genomic region of DNA. Variants include,but are not limited to “pre-mRNA variants” which are transcriptsproduced from the same genomic DNA that differ from other transcriptsproduced from the same genomic DNA in either their start or stopposition and contain both intronic and exonic sequence. Variants alsoinclude, but are not limited to, those with alternate splice junctions,or alternate initiation and termination codons.

As used herein, “high-affinity modified monomer” refers to a monomerhaving at least one modified nucleobase, internucleoside linkage orsugar moiety, when compared to naturally occurring monomers, such thatthe modification increases the affinity of an antisense compoundcomprising the high-affinity modified monomer to its target nucleicacid. High-affinity modifications include, but are not limited to,monomers (e.g., nucleosides and nucleotides) comprising 2′-modifiedsugars.

As used herein, the term “2′-modified” or “2′-substituted” means a sugarcomprising substituent at the 2′ position other than H or OH.2′-modified monomers, include, but are not limited to, BNA's andmonomers (e.g., nucleosides and nucleotides) with 2′-substituents, suchas allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3,O—(CH2)2-O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2-O—N(Rm)(Rn), orO—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H orsubstituted or unsubstituted C1-C10 alkyl. In certain embodiments,oligomeric compounds comprise a 2′ modified monomer that does not havethe formula 2′—O(CH2)nH, wherein n is one to six. In certainembodiments, oligomeric compounds comprise a 2′ modified monomer thatdoes not have the formula 2′-OCH3. In certain embodiments, oligomericcompounds comprise a 2′ modified monomer that does not have the formulaor, in the alternative, 2′—O(CH2)2OCH3.

As used herein, the term “bicyclic nucleic acid” or “BNA” or “bicyclicnucleoside” or “bicyclic nucleotide” refers to a nucleoside ornucleotide wherein the furanose portion of the nucleoside includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

As used herein, unless otherwise indicated, the term “methyleneoxy BNA”alone refers to β-D-methyleneoxy BNA.

As used herein, the term “MOE” refers to a 2′-O-methoxyethylsubstituent.

As used herein, the term “gapmer” refers to a chimeric oligomericcompound comprising a central region (a “gap”) and a region on eitherside of the central region (the “wings”), wherein the gap comprises atleast one modification that is different from that of each wing. Suchmodifications include nucleobase, monomeric linkage, and sugarmodifications as well as the absence of modification (unmodified). Thus,in certain embodiments, the nucleotide linkages in each of the wings aredifferent than the nucleotide linkages in the gap. In certainembodiments, each wing comprises nucleotides with high affinitymodifications and the gap comprises nucleotides that do not comprisethat modification. In certain embodiments the nucleotides in the gap andthe nucleotides in the wings all comprise high affinity modifications,but the high affinity modifications in the gap are different than thehigh affinity modifications in the wings. In certain embodiments, themodifications in the wings are the same as one another. In certainembodiments, the modifications in the wings are different from eachother. In certain embodiments, nucleotides in the gap are unmodified andnucleotides in the wings are modified. In certain embodiments, themodification(s) in each wing are the same. In certain embodiments, themodification(s) in one wing are different from the modification(s) inthe other wing. In certain embodiments, oligomeric compounds are gapmershaving 2′-deoxynucleotides in the gap and nucleotides with high-affinitymodifications in the wing.

As used herein, the term “prodrug” refers to a therapeutic agent that isprepared in an inactive form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts of active compounds that retain the desired biological activity ofthe active compound and do not impart undesired toxicological effectsthereto.

As used herein, the term “cap structure” or “terminal cap moiety” refersto chemical modifications, which have been incorporated at eitherterminus of an antisense compound.

As used herein, the term “prevention” refers to delaying or forestallingthe onset or development of a condition or disease for a period of timefrom hours to days, preferably weeks to months.

As used herein, the term “amelioration” refers to a lessening of atleast one activity or one indicator of the severity of a condition ordisease. The severity of indicators may be determined by subjective orobjective measures which are known to those skilled in the art.

As used herein, the term “treatment” refers to administering acomposition of the invention to effect an alteration or improvement ofthe disease or condition. Prevention, amelioration, and/or treatment mayrequire administration of multiple doses at regular intervals, or priorto onset of the disease or condition to alter the course of the diseaseor condition. Moreover, a single agent may be used in a singleindividual for each prevention, amelioration, and treatment of acondition or disease sequentially, or concurrently.

As used herein, the term “pharmaceutical agent” refers to a substancethat provides a therapeutic benefit when administered to a subject. Incertain embodiments, a pharmaceutical agent is an active pharmaceuticalagent. In certain embodiments, a pharmaceutical agent is a prodrug.

As used herein, the term “therapeutically effective amount” refers to anamount of a pharmaceutical agent that provides a therapeutic benefit toan animal.

As used herein, “administering” means providing a pharmaceutical agentto an animal, and includes, but is not limited to administering by amedical professional and self-administering.

As used herein, the term “co-administering” means providing more thanone pharmaceutical agent to an animal. In certain embodiments, such morethan one pharmaceutical agents are administered together. In certainembodiments, such more than one pharmaceutical agents are administeredseparately. In certain embodiments, such more than one pharmaceuticalagents are administered at the same time. In certain embodiments, suchmore than one pharmaceutical agents are administered at different times.In certain embodiments, such more than one pharmaceutical agents areadministered through the same route of administration. In certainembodiments, such more than one pharmaceutical agents are administeredthrough different routes of administration. In certain embodiments, suchmore than one pharmaceutical agents are contained in the samepharmaceutical formulation. In certain embodiments, such more than onepharmaceutical agents are in separate formulations.

As used herein, the term “pharmaceutical composition” refers to amixture of substances suitable for administering to an individual. Forexample, a pharmaceutical composition may comprise an antisenseoligonucleotide and a sterile aqueous solution. In certain embodiments,a pharmaceutical composition includes a pharmaceutical agent and adiluent and/or carrier.

As used herein, the term “animal” refers to a human or non-human animal,including, but not limited to, mice, rats, rabbits, dogs, cats, pigs,and non-human primates, including, but not limited to, monkeys andchimpanzees.

As used herein, the term “parenteral administration,” refers toadministration through injection or infusion. Parenteral administrationincludes, but is not limited to, subcutaneous administration,intravenous administration, or intramuscular administration.

As used herein, the term “subcutaneous administration” refers toadministration just below the skin. “Intravenous administration” meansadministration into a vein.

As used herein, the term “dose” refers to a specified quantity of apharmaceutical agent provided in a single administration. In certainembodiments, a dose may be administered in two or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired, the desired dose requires a volume not easilyaccommodated by a single injection. In such embodiments, two or moreinjections may be used to achieve the desired dose. In certainembodiments, a dose may be administered in two or more injections tominimize injection site reaction in an individual.

As used herein, the term “dosage unit” refers to a form in which apharmaceutical agent is provided. In certain embodiments, a dosage unitis a vial comprising lyophilized antisense oligonucleotide. In certainembodiments, a dosage unit is a vial comprising reconstituted antisenseoligonucleotide.

As used herein, the term “active pharmaceutical ingredient” refers tothe substance in a pharmaceutical composition that provides a desiredeffect.

As used herein, the term “side effects” refers to physiologicalresponses attributable to a treatment other than desired effects. Incertain embodiments, side effects include, without limitation, injectionsite reactions, liver function test abnormalities, renal functionabnormalities, liver toxicity, renal toxicity, central nervous systemabnormalities, and myopathies. For example, increased aminotransferaselevels in serum may indicate liver toxicity or liver functionabnormality. For example, increased bilirubin may indicate livertoxicity or liver function abnormality.

As used herein, the term “alkyl,” as used herein, refers to a saturatedstraight or branched hydrocarbon radical containing up to twenty fourcarbon atoms. Examples of alkyl groups include, but are not limited to,methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyland the like. Alkyl groups typically include from 1 to about 24 carbonatoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl)with from 1 to about 6 carbon atoms being more preferred. The term“lower alkyl” as used herein includes from 1 to about 6 carbon atoms.Alkyl groups as used herein may optionally include one or more furthersubstituent groups.

As used herein, the term “alkenyl,” as used herein, refers to a straightor branched hydrocarbon chain radical containing up to twenty fourcarbon atoms and having at least one carbon-carbon double bond. Examplesof alkenyl groups include, but are not limited to, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and thelike. Alkenyl groups typically include from 2 to about 24 carbon atoms,more typically from 2 to about 12 carbon atoms with from 2 to about 6carbon atoms being more preferred. Alkenyl groups as used herein mayoptionally include one or more further substituent groups.

As used herein, the term “alkynyl,” as used herein, refers to a straightor branched hydrocarbon radical containing up to twenty four carbonatoms and having at least one carbon-carbon triple bond. Examples ofalkynyl groups include, but are not limited to, ethynyl, 1-propynyl,1-butynyl, and the like. Alkynyl groups typically include from 2 toabout 24 carbon atoms, more typically from 2 to about 12 carbon atomswith from 2 to about 6 carbon atoms being more preferred. Alkynyl groupsas used herein may optionally include one or more further substitutentgroups.

As used herein, the term “aminoalkyl” as used herein, refers to an aminosubstituted alkyl radical. This term is meant to include C1-C12 alkylgroups having an amino substituent at any position and wherein the alkylgroup attaches the aminoalkyl group to the parent molecule. The alkyland/or amino portions of the aminoalkyl group can be further substitutedwith substituent groups.

As used herein, the term “aliphatic,” as used herein, refers to astraight or branched hydrocarbon radical containing up to twenty fourcarbon atoms wherein the saturation between any two carbon atoms is asingle, double or triple bond. An aliphatic group preferably containsfrom 1 to about 24 carbon atoms, more typically from 1 to about 12carbon atoms with from 1 to about 6 carbon atoms being more preferred.The straight or branched chain of an aliphatic group may be interruptedwith one or more heteroatoms that include nitrogen, oxygen, sulfur andphosphorus. Such aliphatic groups interrupted by heteroatoms includewithout limitation polyalkoxys, such as polyalkylene glycols,polyamines, and polyimines. Aliphatic groups as used herein mayoptionally include further substitutent groups.

As used herein, the term “alicyclic” or “alicyclyl” refers to a cyclicring system wherein the ring is aliphatic. The ring system can compriseone or more rings wherein at least one ring is aliphatic. Preferredalicyclics include rings having from about 5 to about 9 carbon atoms inthe ring. Alicyclic as used herein may optionally include furthersubstitutent groups. As used herein, the term “alkoxy,” as used herein,refers to a radical formed between an alkyl group and an oxygen atomwherein the oxygen atom is used to attach the alkoxy group to a parentmolecule. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy,n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as usedherein may optionally include further substitutent groups. As usedherein, the terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

As used herein, the terms “aryl” and “aromatic,” as used herein, referto a mono- or polycyclic carbocyclic ring system radicals having one ormore aromatic rings. Examples of aryl groups include, but are notlimited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl andthe like. Preferred aryl ring systems have from about 5 to about 20carbon atoms in one or more rings. Aryl groups as used herein mayoptionally include further substitutent groups.

As used herein, the terms “aralkyl” and “arylalkyl,” as used herein,refer to a radical formed between an alkyl group and an aryl groupwherein the alkyl group is used to attach the aralkyl group to a parentmolecule. Examples include, but are not limited to, benzyl, phenethyland the like. Aralkyl groups as used herein may optionally includefurther substitutent groups attached to the alkyl, the aryl or bothgroups that form the radical group.

As used herein, the term “heterocyclic radical” as used herein, refersto a radical mono-, or poly-cyclic ring system that includes at leastone heteroatom and is unsaturated, partially saturated or fullysaturated, thereby including heteroaryl groups. Heterocyclic is alsomeant to include fused ring systems wherein one or more of the fusedrings contain at least one heteroatom and the other rings can containone or more heteroatoms or optionally contain no heteroatoms. Aheterocyclic group typically includes at least one atom selected fromsulfur, nitrogen or oxygen. Examples of heterocyclic groups include,[1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as usedherein may optionally include further substitutent groups. As usedherein, the terms “heteroaryl,” and “heteroaromatic,” as used herein,refer to a radical comprising a mono- or poly-cyclic aromatic ring, ringsystem or fused ring system wherein at least one of the rings isaromatic and includes one or more heteroatom. Heteroaryl is also meantto include fused ring systems including systems where one or more of thefused rings contain no heteroatoms. Heteroaryl groups typically includeone ring atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals can be attached to a parent molecule directlyor through a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstitutent groups.

As used herein, the term “heteroarylalkyl,” as used herein, refers to aheteroaryl group as previously defined having an alky radical that canattach the heteroarylalkyl group to a parent molecule. Examples include,but are not limited to, pyridinylmethyl, pyrimidinylethyl,napthyridinylpropyl and the like. Heteroarylalkyl groups as used hereinmay optionally include further substitutent groups on one or both of theheteroaryl or alkyl portions.

As used herein, the term “mono or poly cyclic structure” as used in thepresent invention includes all ring systems that are single orpolycyclic having rings that are fused or linked and is meant to beinclusive of single and mixed ring systems individually selected fromaliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,heterocyclic, heteroaryl, heteroaromatic, heteroarylalkyl. Such mono andpoly cyclic structures can contain rings that are uniform or havevarying degrees of saturation including fully saturated, partiallysaturated or fully unsaturated. Each ring can comprise ring atomsselected from C, N, O and S to give rise to heterocyclic rings as wellas rings comprising only C ring atoms which can be present in a mixedmotif such as for example benzimidazole wherein one ring has only carbonring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic structures can be further substituted with substituent groupssuch as for example phthalimide which has two ═O groups attached to oneof the rings. In another aspect, mono or poly cyclic structures can beattached to a parent molecule directly through a ring atom, through asubstituent group or a bifunctional linking moiety.

As used herein, the term “acyl,” as used herein, refers to a radicalformed by removal of a hydroxyl group from an organic acid and has thegeneral formula —C(O)—X where X is typically aliphatic, alicyclic oraromatic. Examples include aliphatic carbonyls, aromatic carbonyls,aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromaticphosphates, aliphatic phosphates and the like. Acyl groups as usedherein may optionally include further substitutent groups.

As used herein, the term “hydrocarbyl” includes groups comprising C, Oand H. Included are straight, branched and cyclic groups having anydegree of saturation. Such hydrocarbyl groups can include one or moreheteroatoms selected from N, O and S and can be further mono or polysubstituted with one or more substituent groups.

As used herein, the terms “substituent” and “substituent group,” as usedherein, include groups that are typically added to other groups orparent compounds to enhance desired properties or give desired effects.Substituent groups can be protected or unprotected and can be added toone available site or to many available sites in a parent compound.Substituent groups may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound. Such groupsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)Raa), carboxyl (—C(O)O-Raa), aliphatic groups, alicyclicgroups, alkoxy, substituted oxo (—O-Raa), aryl, aralkyl, heterocyclic,heteroaryl, heteroarylalkyl, amino (—NRbbRcc), imino(═NRbb), amido(—C(O)N-RbbRccor —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano(—CN), carbamido (—OC(O)NRbbRcc or —N(Rbb)C(O)ORaa), ureido(—N(Rbb)C(O)NRbbRcc), thioureido (—N(Rbb)C(S)NRbbRcc), guanidinyl(—N(Rbb)C(═NRbb)NRbbRcc), amidinyl (—C(═NRbb)-NRbbRcc or—N(Rbb)C(NRbb)Raa), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl(—S(O)2Rbb), sulfonamidyl (—S(O)2NRbbRcc or —N(Rbb)S(O)2Rbb) andconjugate groups. Wherein each Raa, Rbb and Rcc is, independently, H, anoptionally linked chemical functional group or a further substituentgroup with a preferred list including without limitation H, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl.

Oligomeric Compounds

Antisense compounds and antidote compounds are oligomeric compounds. Incertain embodiments, it is desirable to chemically modify oligomericcompounds, including antisense compounds and/or antidote oligomericcompounds, compared to naturally occurring oligomers, such as DNA orRNA. Certain such modifications alter the activity of the oligomericcompound. Certain such chemical modifications can alter activity by, forexample: increasing affinity of an antisense compound for its targetnucleic acid or an antidote for its antisense compound, increasing itsresistance to one or more nucleases, and/or altering thepharmacokinetics or tissue distribution of the oligomeric compound. Incertain instances, the use of chemistries that increase the affinity ofan oligomeric compound for its target can allow for the use of shorteroligomeric compounds.

Certain Monomers

In certain embodiment, oligomeric compounds comprise one or moremodified monomer. In certain such embodiments, oligomeric compoundscomprise one or more high affinity monomer. In certain embodiments, suchhigh-affinity monomer is selected from monomers (e.g., nucleosides andnucleotides) comprising 2′-modified sugars, including, but not limitedto: BNA's and monomers (e.g., nucleosides and nucleotides) with2′-substituents such as allyl, amino, azido, thio, O-allyl, O—C1-C10alkyl, —OCF3, O—(CH2)2-O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2-O—N(Rm)(Rn), orO—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H orsubstituted or unsubstituted C1-C10 alkyl.

In certain embodiments, the oligomeric compounds including, but nolimited to antidote and antisense oligomeric compounds of the presentinvention, comprise one or more high affinity monomers provided that theoligomeric compound does not comprise a nucleotide comprising a2′-O(CH2)nH, wherein n is one to six.

In certain embodiments, the oligomeric compounds including, but nolimited to antidote and antisense oligomeric compounds, comprise one ormore high affinity monomer provided that the oligomeric compound doesnot comprise a nucleotide comprising a 2′-OCH3 or a 2′—O(CH2)2OCH3.

In certain embodiments, the oligomeric compounds including, but nolimited to antidote and antisense oligomeric compounds, comprise one ormore high affinity monomer provided that the oligomeric compound doesnot comprise a α-L-Methyleneoxy (4′-CH2-O-2′) BNA.

In certain embodiments, the oligomeric compounds including, but nolimited to antidote and antisense oligomeric compounds, comprise one ormore high affinity monomer provided that the oligomeric compound doesnot comprise a β-D-Methyleneoxy (4′-CH2-O-2′) BNA.

In certain embodiments, the oligomeric compounds including, but nolimited to antidote and antisense oligomeric compounds, comprise one ormore high affinity monomer provided that the oligomeric compound doesnot comprise a α-L-Methyleneoxy (4′-CH2-O-2′) BNA or a 13-D-Methyleneoxy(4′-CH2-O-2′) BNA.

Certain Nucleobases

The naturally occurring base portion of a nucleoside is typically aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. For those nucleosides thatinclude a pentofuranosyl sugar, a phosphate group can be linked to the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,those phosphate groups covalently link adjacent nucleosides to oneanother to form a linear polymeric compound. Within oligonucleotides,the phosphate groups are commonly referred to as forming theinternucleoside backbone of the oligonucleotide. The naturally occurringlinkage or backbone of RNA and of DNA is a 3′ to 5′ phosphodiesterlinkage.

In addition to “unmodified” or “natural” nucleobases such as the purinenucleobases adenine (A) and guanine (G), and the pyrimidine nucleobasesthymine (T), cytosine (C) and uracil (U), many modified nucleobases ornucleobase mimetics known to those skilled in the art are amenable withthe compounds described herein. In certain embodiments, a modifiednucleobase is a nucleobase that is fairly similar in structure to theparent nucleobase, such as for example a 7-deaza purine, a 5-methylcytosine, or a G-clamp. In certain embodiments, nucleobase mimeticinclude more complicated structures, such as for example a tricyclicphenoxazine nucleobase mimetic. Methods for preparation of the abovenoted modified nucleobases are well known to those skilled in the art.

Certain Sugars

Oligomeric compounds provided herein may comprise one or more monomer,including a nucleoside or nucleotide, having a modified sugar moiety.For example, the furanosyl sugar ring of a nucleoside can be modified ina number of ways including, but not limited to, addition of asubstituent group, bridging of two non-geminal ring atoms to form abicyclic nucleic acid (BNA). In certain embodiments, oligomericcompounds comprise one or more monomers that is a BNA. In certain suchembodiments, BNA s include, but are not limited to, (A) α-L-Methyleneoxy(4′-CH2-O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH2-O-2′) BNA, (C)Ethyleneoxy (4′-(CH2)2-O-2′) BNA, (D) Aminooxy (4′-CH2-O—N(R)-2′) BNAand (E) Oxyamino (4′-CH2-N(R)—O-2′) BNA, as depicted below:

In certain embodiments, BNA compounds include, but are not limited to,compounds having at least one bridge between the 4′ and the 2′ positionof the sugar wherein each of the bridges independently comprises 1 orfrom 2 to 4 linked groups independently selected from —[C(R1)(R2)]n—,—C(R1)=C(R2)-, —C(R1)=N—, —C(═NR1)-, —C(═O)—, —C(═S)—, —O—, —Si(R1)2-,—S(═O)x- and —N(R1)-;

wherein:

x is 0, 1, or 2;n is 1, 2, 3, or 4;

each R1 and R2 is, independently, H, a protecting group, hydroxyl,C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substitutedC2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl,substituted C5-C20 aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical,substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3,COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), orsulfoxyl (S(═O)-J1); and

each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl,substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl ora protecting group.

In one embodiment, each of the bridges of the BNA compounds is,independently, —[C(R1)(R2)]n-, —[C(R1)(R2)]n-O—, —C(R1R2)-N(R1)-O— or—C(R1R2)-O—N(R1)-. In another embodiment, each of said bridges is,independently, 4′-CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)3-2′, 4′-CH2-O-2′,4′-(CH2)2-O-2′, 4′-CH2-O—N(R1)-2′ and 4′-CH2-N(R1)-O-2′- wherein each R1is, independently, H, a protecting group or C1-C12 alkyl.

Certain BNA's have been prepared and disclosed in the patent literatureas well as in scientific literature (Singh et al., Chem. Commun., 1998,4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedtet al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar etal., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94/14226; WO2005/021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Examples of issued US patents and published applications that discloseBNA s include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490;6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-GrantPublication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841;2004-0143114; and 20030082807.

Also provided herein are BNAs in which the 2′-hydroxyl group of theribosyl sugar ring is linked to the 4′ carbon atom of the sugar ringthereby forming a methyleneoxy (4′-CH2-O-2′) linkage to form thebicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens.Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; andOrum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S.Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene(—CH2-) group bridging the 2′ oxygen atom and the 4′ carbon atom, forwhich the term methyleneoxy (4′-CH2-O-2′) BNA is used for the bicyclicmoiety; in the case of an ethylene group in this position, the termethyleneoxy (4′-CH2CH2-O-2′) BNA is used (Singh et al., Chem. Commun.,1998, 4, 455-456: Morita et al., Bioorganic Medicinal Chemistry, 2003,11, 2211-2226). Methyleneoxy (4′-CH2-O-2′) BNA and other bicyclic sugaranalogs display very high duplex thermal stabilities with complementaryDNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolyticdegradation and good solubility properties. Potent and nontoxicantisense oligonucleotides comprising BNAs have been described(Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

An isomer of methyleneoxy (4′-CH2-O-2′) BNA that has also been discussedis alpha-L-methyleneoxy (4′-CH2-O-2′) BNA which has been shown to havesuperior stability against a 3′-exonuclease. The alpha-L-methyleneoxy(4′-CH2-O-2′) BNA's were incorporated into antisense gapmers andchimeras that showed potent antisense activity (Frieden et al., NucleicAcids Research, 2003, 21, 6365-6372).

The synthesis and preparation of the methyleneoxy (4′-CH2-O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH2-O-2′) BNA, phosphorothioate-methyleneoxy(4′-CH2-O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar etal., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation oflocked nucleoside analogs comprising oligodeoxyribonucleotide duplexesas substrates for nucleic acid polymerases has also been described(Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, anovel comformationally restricted high-affinity oligonucleotide analoghas been described in the art (Singh et al., J. Org. Chem., 1998, 63,10035-10039). In addition, 2′-Amino- and 2′-methylamino-BNA's have beenprepared and the thermal stability of their duplexes with complementaryRNA and DNA strands has been previously reported.

Modified sugar moieties are well known and can be used to alter,typically increase, the affinity of the antisense compound for itstarget and/or increase nuclease resistance. A representative list ofpreferred modified sugars includes but is not limited to bicyclicmodified sugars (BNA's), including methyleneoxy (4′-CH2-O-2′) BNA andethyleneoxy (4′-(CH2)2-O-2′ bridge) BNA; substituted sugars, especially2′-substituted sugars having a 2′-F, 2′-OCH3 or a 2′—O(CH2)2-OCH3substituent group; and 4′-thio modified sugars. Sugars can also bereplaced with sugar mimetic groups among others. Methods for thepreparations of modified sugars are well known to those skilled in theart. Some representative patents and publications that teach thepreparation of such modified sugars include, but are not limited to,U.S. Patents: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032;and WO 2005/121371.

In certain embodiments, BNA's include bicyclic nucleoside having theformula:

wherein:

Bx is a heterocyclic base moiety;

T1 is H or a hydroxyl protecting group;

T2 is H, a hydroxyl protecting group or a reactive phosphorus group;

Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl,substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substitutedacyl, or substituted amide.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with optionally protected substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3,OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein each J1, J2 and J3is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.

In certain such embodiments, each of the substituted groups, is,independently, mono or poly substituted with substituent groupsindependently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3,OC(═X)J1, and NJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is,independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O orNJ1. In certain embodiments, the Z group is C1-C6 alkyl substituted withone or more Xx, wherein each Xx is independently OJ1, NJ1J2, SJ1, N3,OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In anotherembodiment, the Z group is C1-C6 alkyl substituted with one or more Xx,wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy(e.g., CH3O—), substituted alkoxy or azido.

In certain embodiments, the Z group is —CH2Xx, wherein Xx is OJ1, NJ1J2,SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. Inanother embodiment, the Z group is —CH2Xx, wherein Xx is halo (e.g.,fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

In certain such embodiments, the Z group is in the (R)-configuration:

In certain such embodiments, the Z group is in the (S)-configuration:

In certain embodiments, each T1 and T2 is a hydroxyl protecting group. Apreferred list of hydroxyl protecting groups includes benzyl, benzoyl,2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl)and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, T1is a hydroxyl protecting group selected from acetyl, benzyl,t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein amore preferred hydroxyl protecting group is T1 is 4,4′-dimethoxytrityl.

In certain embodiments, T2 is a reactive phosphorus group whereinpreferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In certain embodiments T1 is4,4′-dimethoxytrityl and T2 is diisopropylcyanoethoxy phosphoramidite.

In certain embodiments, oligomeric compounds have at least one monomerof the formula:

or of the formula:

or of the formula:

wherein

Bx is a heterocyclic base moiety;

T3 is H, a hydroxyl protecting group, a linked conjugate group or aninternucleoside linking group attached to a nucleoside, a nucleotide, anoligonucleoside, an oligonucleotide, a monomeric subunit or anoligomeric compound;

T4 is H, a hydroxyl protecting group, a linked conjugate group or aninternucleoside linking group attached to a nucleoside, a nucleotide, anoligonucleoside, an oligonucleotide, a monomeric subunit or anoligomeric compound;

wherein at least one of T3 and T4 is an internucleoside linking groupattached to a nucleoside, a nucleotide, an oligonucleoside, anoligonucleotide, a monomeric subunit or an oligomeric compound; and

Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl,substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substitutedacyl, or substituted amide. In one embodiment, each of the substitutedgroups, is, independently, mono or poly substituted with optionallyprotected substituent groups independently selected from halogen, oxo,hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 andCN, wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, andX is O, S or NJ1.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, andNJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is, independently, H or C1-C6alkyl, and X is O or NJ1.

In certain such embodiments, at least one Z is C1-C6 alkyl orsubstituted C1-C6 alkyl. In certain embodiments, each Z is,independently, C1-C6 alkyl or substituted C1-C6 alkyl. In certainembodiments, at least one Z is C1-C6 alkyl. In certain embodiments, eachZ is, independently, C1-C6 alkyl. In certain embodiments, at least one Zis methyl. In certain embodiments, each Z is methyl. In certainembodiments, at least one Z is ethyl. In certain embodiments, each Z isethyl. In certain embodiments, at least one Z is substituted C1-C6alkyl. In certain embodiments, each Z is, independently, substitutedC1-C6 alkyl. In certain embodiments, at least one Z is substitutedmethyl. In certain embodiments, each Z is substituted methyl. In certainembodiments, at least one Z is substituted ethyl. In certainembodiments, each Z is substituted ethyl.

In certain embodiments, at least one substituent group is C1-C6 alkoxy(e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6alkoxy). In another embodiment, each substituent group is,independently, C1-C6 alkoxy (e.g., each Z is, independently, C1-C6 alkylsubstituted with one or more C1-C6 alkoxy).

In certain embodiments, at least one C1-C6 alkoxy substituent group isCH3O— (e.g., at least one Z is CH3OCH2-). In another embodiment, eachC1-C6 alkoxy substituent group is CH3O— (e.g., each Z is CH3OCH2-).

In certain embodiments, at least one substituent group is halogen (e.g.,at least one Z is C1-C6 alkyl substituted with one or more halogen). Incertain embodiments, each substituent group is, independently, halogen(e.g., each Z is, independently, C1-C6 alkyl substituted with one ormore halogen). In certain embodiments, at least one halogen substituentgroup is fluoro (e.g., at least one Z is CH2FCH2-, CHF2CH2- or CF3CH2-).In certain embodiments, each halo substituent group is fluoro (e.g.,each Z is, independently, CH2FCH2-, CHF2CH2- or CF3CH2-).

In certain embodiments, at least one substituent group is hydroxyl(e.g., at least one Z is C1-C6 alkyl substituted with one or morehydroxyl). In certain embodiments, each substituent group is,independently, hydroxyl (e.g., each Z is, independently, C1-C6 alkylsubstituted with one or more hydroxyl). In certain embodiments, at leastone Z is HOCH2-. In another embodiment, each Z is HOCH2-.

In certain embodiments, at least one Z is CH3-, CH3CH2-, CH2OCH3-, CH2F—or HOCH2-. In certain embodiments, each Z is, independently, CH3-,CH3CH2-, CH2OCH3-, CH2F— or HOCH2-.

In certain embodiments, at least one Z group is C1-C6 alkyl substitutedwith one or more Xx, wherein each Xx is, independently, OJ1, NJ1J2, SJ1,N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 andJ3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In anotherembodiment, at least one Z group is C1-C6 alkyl substituted with one ormore Xx, wherein each Xx is, independently, halo (e.g., fluoro),hydroxyl, alkoxy (e.g., CH3O—) or azido.

In certain embodiments, each Z group is, independently, C1-C6 alkylsubstituted with one or more Xx, wherein each Xx is independently OJ1,NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein eachJ1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.In another embodiment, each Z group is, independently, C1-C6 alkylsubstituted with one or more Xx, wherein each Xx is independently halo(e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

In certain embodiments, at least one Z group is —CH2Xx, wherein Xx isOJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; whereineach J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S orNJ1 In certain embodiments, at least one Z group is —CH2Xx, wherein Xxis halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

In certain embodiments, each Z group is, independently, —CH2Xx, whereineach Xx is, independently, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2,NJ3C(═X)NJ1J2 or CN;

wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X isO, S or NJ1. In another embodiment, each Z group is, independently,—CH2Xx, wherein each Xx is, independently, halo (e.g., fluoro),hydroxyl, alkoxy (e.g., CH3O—) or azido. In certain embodiments, atleast one Z is CH3-. In another embodiment, each Z is, CH3-.

In certain embodiments, the Z group of at least one monomer is in the(R)-configuration represented by the formula:

or the formula:

or the formula:

In certain embodiments, the Z group of each monomer of the formula is inthe (R)-configuration.

In certain embodiments, the Z group of at least one monomer is in the(S)-configuration represented by the formula:

or the formula:

or the formula:

In certain embodiments, the Z group of each monomer of the formula is inthe (S)-configuration.

In certain embodiments, T3 is H or a hydroxyl protecting group. Incertain embodiments, T4 is H or a hydroxyl protecting group. In afurther embodiment T3 is an internucleoside linking group attached to anucleoside, a nucleotide or a monomeric subunit. In certain embodiments,T4 is an internucleoside linking group attached to a nucleoside, anucleotide or a monomeric subunit. In certain embodiments, T3 is aninternucleoside linking group attached to an oligonucleoside or anoligonucleotide. In certain embodiments, T4 is an internucleosidelinking group attached to an oligonucleoside or an oligonucleotide. Incertain embodiments, T3 is an internucleoside linking group attached toan oligomeric compound. In certain embodiments, T4 is an internucleosidelinking group attached to an oligomeric compound. In certainembodiments, at least one of T3 and T4 comprises an internucleosidelinking group selected from phosphodiester or phosphorothioate.

In certain embodiments, oligomeric compounds have at least one region ofat least two contiguous monomers of the formula:

or of the formula:

or of the formula:

to

In certain embodiments, the oligomeric compound comprises at least tworegions of at least two contiguous monomers of the above formula. Incertain embodiments, the oligomeric compound comprises a gappedoligomeric compound. In certain embodiments, the oligomeric compoundcomprises at least one region of from about 8 to about 14 contiguousβ-D-2′-deoxyribofuranosyl nucleosides. In certain embodiments, theoligomeric compound comprises at least one region of from about 9 toabout 12 contiguous β-D-2′-deoxyribofuranosyl nucleosides.

In certain embodiments, monomers include sugar mimetics. In certain suchembodiments, a mimetic is used in place of the sugar orsugar-internucleoside linkage combination, and the nucleobase ismaintained for hybridization to a selected target. Representativeexamples of a sugar mimetics include, but are not limited to,cyclohexenyl or morpholino. Representative examples of a mimetic for asugar-internucleoside linkage combination include, but are not limitedto, peptide nucleic acids (PNA) and morpholino groups linked byuncharged achiral linkages. In some instances a mimetic is used in placeof the nucleobase. Representative nucleobase mimetics are well known inthe art and include, but are not limited to, tricyclic phenoxazineanalogs and universal bases (Berger et al., Nuc Acid Res. 2000,28:2911-14, incorporated herein by reference). Methods of synthesis ofsugar, nucleoside and nucleobase mimetics are well known to thoseskilled in the art.

Monomeric Linkages

Described herein are linking groups that link monomers (including, butnot limited to, modified and unmodified nucleosides and nucleotides)together, thereby forming an oligomeric compound. The two main classesof linking groups are defined by the presence or absence of a phosphorusatom. Representative phosphorus containing linkages include, but are notlimited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing linking groups include, but are not limitedto, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—),thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); andN,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-). Oligomeric compoundshaving non-phosphorus linking groups are referred to asoligonucleosides. Modified linkages, compared to natural phosphodiesterlinkages, can be used to alter, typically increase, nuclease resistanceof the oligomeric compound. In certain embodiments, linkages having achiral atom can be prepared a racemic mixtures, as separate enantomers.Representative chiral linkages include, but are not limited to,alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing linkages are wellknown to those skilled in the art.

The oligomeric compounds described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or(L) such as for amino acids et al. Included in the antisense compoundsprovided herein are all such possible isomers, as well as their racemicand optically pure forms.

Oligomeric Compounds

In certain embodiments, provided herein are oligomeric compounds havingreactive phosphorus groups useful for forming linkages including forexample phosphodiester and phosphorothioate internucleoside linkages.Methods of preparation and/or purification of precursors or oligomericcompounds are not a limitation of the compositions or methods providedherein. Methods for synthesis and purification of oligomeric compoundsincluding DNA, RNA, oligonucleotides, oligonucleosides, and antisensecompounds are well known to those skilled in the art.

Generally, oligomeric compounds comprise a plurality of monomericsubunits linked together by linking groups. Nonlimiting examples ofoligomeric compounds include primers, probes, antisense compounds,antisense oligonucleotides, external guide sequence (EGS)oligonucleotides, alternate splicers, and siRNAs. As such, thesecompounds can be introduced in the form of single-stranded,double-stranded, circular, branched or hairpins and can containstructural elements such as internal or terminal bulges or loops.Oligomeric double-stranded compounds can be two strands hybridized toform double-stranded compounds or a single strand with sufficient selfcomplementarity to allow for hybridization and formation of a fully orpartially double-stranded compound.

In certain embodiments, the present invention provides chimericoligomeric compounds. In certain such embodiments, chimeric oligomericcompounds are chimeric oligonucleotides. In certain such embodiments,the chimeric oligonucleotides comprise differently modified nucleotides.In certain embodiments, chimeric oligonucleotides are mixed-backboneantisense oligonucleotides.

In general a chimeric oligomeric compound will have modified nucleosidesthat can be in isolated positions or grouped together in regions thatwill define a particular motif Any combination of modifications and/ormimetic groups can comprise a chimeric oligomeric compound as describedherein.

In certain embodiments, chimeric oligomeric compounds typically compriseat least one region modified so as to confer increased resistance tonuclease degradation, increased cellular uptake, and/or increasedbinding affinity for the target nucleic acid. In certain embodiments, anadditional region of the oligomeric compound may serve as a substratefor enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.

In certain embodiments, chimeric oligomeric compounds are gapmers. Incertain such embodiments, a mixed-backbone oligomeric compound has onetype of internucleotide linkages in one or both wings and a differenttype of internucleoside linkages in the gap. In certain suchembodiments, the mixed-backbone oligonucleotide has phosphodiesterlinkages in the wings and phosphorothioate linkages in the gap. Incertain embodiments in which the internucleoside linkages in a wing isdifferent from the internucleoside linkages in the gap, theinternucleoside linkage bridging that wing and the gap is the same asthe internucleoside linkage in the wing. In certain embodiments in whichthe internucleoside linkages in a wing is different from theinternucleoside linkages in the gap, the internucleoside linkagebridging that wing and the gap is the same as the internucleosidelinkage in the gap.

In certain embodiments, the present invention provides oligomericcompounds, including antisense oligomeric compounds and antidoteoligomeric compounds, of any of a variety of ranges of lengths. Incertain embodiments, the invention provides oligomeric compoundsconsisting of X-Y linked oligonucleosides, where X and Y are eachindependently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; providedthat X<Y. For example, in certain embodiments, the invention providesoligomeric compounds comprising: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14,8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26,8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17,9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29,9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19,10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28, 10-29,10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20,11-21, 11-22, 11-23, 11-24, 11-25, 11-26, 11-27, 11-28, 11-29, 11-30,12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22,12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14, 13-15,13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25,13-26, 13-27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19,14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29,14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24,15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19, 16-20,16-21, 16-22, 16-23, 16-24, 16-25, 26, 16-27, 16-28, 16-29, 16-30,17-18, 17-19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27,17-28, 17-29, 17-30, 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25,18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19-21, 19-22, 19-23, 19-24,19-25, 19-26, 19-29, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24,20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25,21-26, 21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, 22-27,22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30,24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27, 25-28, 25-29,25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29, 28-30, or29-30 linked nucleosides.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by covalentattachment of one or more conjugate groups. In general, conjugate groupsmodify one or more properties of the attached oligomeric compoundincluding but not limited to pharmacodynamic, pharmacokinetic, binding,absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional linking moiety or linking groupto a parent compound such as an oligomeric compound. A preferred list ofconjugate groups includes without limitation, intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, thioethers,polyethers, cholesterols, thiocholesterols, cholic acid moieties,folate, lipids, phospholipids, biotin, phenazine, phenanthridine,anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarinsand dyes.

Preferred conjugate groups amenable to the present invention includelipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al.,Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49); a phospholipid, e.g.,di-hexadecyl-rac-glycerol ortriethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl.Acids Res., 1990, 18, 3777); a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); apalmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264,229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety(Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Linking groups or bifunctional linking moieties such as those known inthe art are amenable to the compounds provided herein. Linking groupsare useful for attachment of chemical functional groups, conjugategroups, reporter groups and other groups to selective sites in a parentcompound such as for example an oligomeric compound. In general abifunctional linking moiety comprises a hydrocarbyl moiety having twofunctional groups. One of the functional groups is selected to bind to aparent molecule or compound of interest and the other is selected tobind essentially any selected group such as chemical functional group ora conjugate group. In some embodiments, the linker comprises a chainstructure or an oligomer of repeating units such as ethylene glycol oramino acid units. Examples of functional groups that are routinely usedin a bifunctional linking moiety include, but are not limited to,electrophiles for reacting with nucleophilic groups and nucleophiles forreacting with electrophilic groups. In some embodiments, bifunctionallinking moieties include amino, hydroxyl, carboxylic acid, thiol,unsaturations (e.g., double or triple bonds), and the like. Somenonlimiting examples of bifunctional linking moieties include8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C1-C10 alkyl, substituted orunsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Synthesis, Purification and Analysis

Oligomerization of modified and unmodified nucleosides and nucleotidescan be routinely performed according to literature procedures for DNA(Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), HumanaPress) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al.,Applications of Chemically synthesized RNA in RNA: Protein Interactions,Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57,5707-5713).

Oligomeric compounds provided herein can be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed. It is well known to use similar techniques to prepareoligonucleotides such as the phosphorothioates and alkylatedderivatives. The invention is not limited by the method of antisensecompound synthesis.

Methods of purification and analysis of oligomeric compounds are knownto those skilled in the art. Analysis methods include capillaryelectrophoresis (CE) and electrospray-mass spectroscopy. Such synthesisand analysis methods can be performed in multi-well plates. The methodof the invention is not limited by the method of oligomer purification.

Compositions and Methods for Formulating Pharmaceutical Compositions

Oligomeric compounds may be admixed with pharmaceutically acceptableactive and/or inert substances for the preparation of pharmaceuticalcompositions or formulations. Compositions and methods for theformulation of pharmaceutical compositions are dependent upon a numberof criteria, including, but not limited to, route of administration,extent of disease, or dose to be administered.

Oligomeric compounds, including antisense compounds and/or antidotecompounds, can be utilized in pharmaceutical compositions by combiningsuch oligomeric compounds with a suitable pharmaceutically acceptablediluent or carrier. A pharmaceutically acceptable diluent includesphosphate-buffered saline (PBS). PBS is a diluent suitable for use incompositions to be delivered parenterally. Accordingly, in oneembodiment, employed in the methods described herein is a pharmaceuticalcomposition comprising an antisense compound and/or antidote compoundand a pharmaceutically acceptable diluent. In certain embodiments, thepharmaceutically acceptable diluent is PBS.

Pharmaceutical compositions comprising oligomeric compounds encompassany pharmaceutically acceptable salts, esters, or salts of such esters.In certain embodiments, pharmaceutical compositions comprisingoligomeric compounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligomeric compound which are cleaved by endogenousnucleases within the body, to form the active oligomeric compound.

Antisense

Antisense mechanisms are all those involving the hybridization of acompound with target nucleic acid, wherein the outcome or effect of thehybridization is either target degradation or target occupancy withconcomitant stalling of the cellular machinery involving, for example,transcription or splicing.

For example, a type of antisense mechanism involving target degradationincludes an RNase H. RNase H is a cellular endonuclease which cleavesthe RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNAse Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

In certain embodiments, chemically-modified antisense compounds have ahigher affinity for target RNAs than does non-modified DNA. In certainsuch embodiments, that higher affinity in turn provides increasedpotency allowing for the administration of lower doses of suchcompounds, reduced potential for toxicity and improvement in therapeuticindex and decreased overall cost of therapy.

Antisense compounds are oligomeric compounds. Accordingly, in certainembodiments, antisense compounds comprise, for example and withoutlimitation, any of the modifications and motifs described in thediscussion above for oligomeric compounds. Antisense compounds may besingle-stranded or double-stranded oligomeric compounds. In embodimentswhere an antisense compound is a double-stranded oligomeric compound,the two strands may have the same modifications and motifs or may havemodifications and motifs that are different from one another. Certainantisense compounds and modifications and motifs useful for suchcompounds are known in the art.

Modulation of Target Expression

In certain embodiments, a target nucleic acid is a mRNA. In certain suchembodiments, antisense compounds are designed to modulate that targetmRNA or its expression. In certain embodiments, designing an antisensecompound to a target nucleic acid molecule can be a multistep process.Typically the process begins with the identification of a targetprotein, the activity of which is to be modulated, and then identifyingthe nucleic acid the expression of which yields the target protein. Incertain embodiments, designing of an antisense compound results in anantisense compound that is hybridizable to the targeted nucleic acidmolecule. In certain embodiments, the antisense compound is an antisenseoligonucleotide or antisense oligonucleoside. In certain embodiments, anantisense compound and a target nucleic acid are complementary to oneanother. In certain such embodiments, an antisense compound is perfectlycomplementary to a target nucleic acid. In certain embodiments, anantisense compound includes one mismatch. In certain embodiments, anantisense compound includes two mismatches. In certain embodiments, anantisense compound includes three or more mismatches.

Modulation of expression of a target nucleic acid can be achievedthrough alteration of any number of nucleic acid functions. In certainembodiments, the functions of RNA to be modulated include, but are notlimited to, translocation functions, which include, but are not limitedto, translocation of the RNA to a site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, and translation of protein from the RNA. RNAprocessing functions that can be modulated include, but are not limitedto, splicing of the RNA to yield one or more RNA species, capping of theRNA, 3′ maturation of the RNA and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. Modulation of expression can result in the increased level ofone or more nucleic acid species or the decreased level of one or morenucleic acid species, either temporally or by net steady state level.Thus, in one embodiment modulation of expression can mean increase ordecrease in target RNA or protein levels. In another embodimentmodulation of expression can mean an increase or decrease of one or moreRNA splice products, or a change in the ratio of two or more spliceproducts.

Hybridization

In certain embodiments, antisense compounds specifically hybridize whenthere is a sufficient degree of complementarity to avoid non-specificbinding of the antisense compound to non-target nucleic acid sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

As used herein, “stringent hybridization conditions” or “stringentconditions” refers to conditions under which an antisense compound willhybridize to its target sequence, but to a minimal number of othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances, and “stringent conditions” underwhich antisense compounds hybridize to a target sequence are determinedby the nature and composition of the antisense compounds and the assaysin which they are being investigated.

It is understood in the art that incorporation of nucleotide affinitymodifications may allow for a greater number of mismatches compared toan unmodified compound. Similarly, certain oligonucleotide sequences maybe more tolerant to mismatches than other oligonucleotide sequences. Oneof ordinary skill in the art is capable of determining an appropriatenumber of mismatches between oligonucleotides, or between anoligonucleotide and a target nucleic acid, such as by determiningmelting temperature (Tm). Tm or ΔTm can be calculated by techniques thatare familiar to one of ordinary skill in the art. For example,techniques described in Freier et al. (Nucleic Acids Research, 1997, 25,22: 4429-4443) allow one of ordinary skill in the art to evaluatenucleotide modifications for their ability to increase the meltingtemperature of an RNA:DNA duplex.

Antidote

In certain instances it is desirable to inhibit antisense activity. Forexample, in certain embodiments where the antisense target is an mRNA,it is may be desirable to inhibit antisense activity and thereby restoreexpression of a target protein. For example, certain antisense compoundshave been used therapeutically. In certain such uses, antisensecompounds are long-acting. In certain instances, such long actingantisense compounds are desirable, for their convenience. In suchinstances, though, it may also be desirable to have a means to reversethe antisense activity of an antisense compound. For example, a patientmay respond poorly to treatment or receive too high a dose. In such aninstance, an antidote to the antisense compound may be administered toat least partially reduce the antisense activity of the antisensecompound. In certain embodiments, the long-lasting effect of antisensecompounds makes waiting for that effect to slowly diminish throughnatural clearance an unattractive option.

By way of example, and without limiting the present invention, certainantisense compounds are useful for inhibiting blood clotting factors(e.g., Factor II (prothrombin), Factor VII, Factor IX, etc.). Certainsuch antisense compounds may be found, for example in Provisional U.S.Application 60/980,376, which is hereby incorporated by reference in itsentirety. Such antisense compounds have therapeutic potential asanticoagulants. Long half-lives make such antisense compoundsparticularly attractive, however, if a patient receives too high a dose,has surgery (where anti-coagulation is undesirable) or otherwise desiresa decrease in the anti-coagulant effect, an antidote to the antisenseanti-coagulant compound may be administered. Such antidote compound willrestore coagulation function more quickly than simply waiting fornatural clearance of the antisense compound. This example is providedfor illustrative purposes. Antisense compounds have been designed to avast number of targets, including without limitation, a vast number ofmessenger RNA (mRNA) targets and pre-mRNA targets, as well as a vastnumber of non-coding RNA targets. Antidotes provided herein are suitablefor any antisense compound, regardless of the target or mechanism of theantisense compound.

In certain embodiments, the invention provides antidote compounds to anantisense compound targeted to an mRNA. In certain such embodiments, thetarget mRNA encodes a protein involved in metabolism. In certain suchembodiments, the target mRNA encodes a protein involved in cardiacfunction. In certain embodiments, the target mRNA encodes a proteininvolved in blood-clotting. Antisense compounds targeting any of avariety of target proteins are known in the art. See, for example:Provisional U.S. Application 60/980,376; U.S. application Ser. No.11/745,429, each of which is hereby incorporated by reference in itsentirety. Target mRNAs that may be modulated with antisense and thenwith an antidote compound include, but are not limited to those encodingany of the following: prothrombin (Factor II), Factor VII, Factor IX,Factor XI, ApoB, SGLT2, PTEN, SOD1, Huntingtin, PTP1B, ICAM-1, CRP,GCGR, GCCR, Clusterin, Survivin, elf-4-e, Hsp27, VLA-4, PCSK-9, DGAT2,and IL-4α.

In certain embodiments, the invention provides antidote compound to anantisense compound that modulates splicing of a pre-mRNA. Certain suchantisense compounds may be found for example in U.S. Pat. Nos.6,172,216; and 6,210,892; in U.S. application Ser. Nos. 10/672,501;11/339,785; and 10/416,214; and in International Application Nos.: WO2007/002390; WO 2007/028065; WO 2007/047913; each of which is herebyincorporated by reference in its entirety.

In certain embodiments, the invention provides antidote compound to anantisense compound that modulates a micro-RNA. Certain such antisensecompounds may be found for example in U.S. application Ser. No.10/909,125; International Application Nos.: WO03/029459, which is herebyincorporated by reference in its entirety.

Antisense activity may rely on any of variety of different mechanisms toexert an effect. For example, a particular antisense compound mayfunction through RNase H cleavage, through the RISC pathway, and/or byblocking translation or altering splicing by simply occupying a targetRNA. Antidote compounds may be designed to any antisense compound,regardless of the mechanism(s) of action of the antisense compound.Likewise, the antidote itself may work through any mechanism(s). Forexample, in certain embodiments, hybridization of the antidote compoundto the antisense compound results in cleavage of the antisense compound.In certain such embodiments, cleavage is affected by RNase H. In certainembodiments, hybridization of the antidote to the antisense compoundsdoes not result in cleavage of the antisense compound, but nonethelessreduces antisense activity.

In certain embodiments, because the antidote compound is in competitionwith the antisense target for binding with the antisense compound, it isdesirable to modify the antidote compound to increase its affinity forthe antisense compound. In certain embodiments, one or more nucleosideof the antidote compound is modified. In certain such embodiments, suchmodification increases the affinity of the antidote compound for theantisense compound. Such modifications are known in the art and include,but are not limited to, BNA, including, but not limited to LNA, and ENA,2′ substitutions including, but not limited to 2′ MOE, 2′-F, 2′-O-alkyl,including, but not limited to 2′-OMe. Such modifications may be used inany combination. In certain embodiments, an antidote is an oligomericcompound. Such antidotes may comprise any modification or motif,including, but not limited to those discussed above and in thereferences cited herein.

Antidote compounds are oligomeric compounds. Accordingly, in certainembodiments, antidote compounds comprise, for example and withoutlimitation, any of the modifications and motifs described in thediscussion above for oligomeric compounds. Antisense compounds may besingle-stranded or double-stranded oligomeric compounds. In embodimentswhere an antisense compound is a double-stranded oligomeric compound,the two strands may have the same modifications and motifs or may havemodifications and motifs that are different from one another. Certainantisense compounds and modifications and motifs useful for suchcompounds are known in the art. Such modifications and motifs maylikewise be useful for antidote compounds.

In certain embodiments, motifs are designed with consideration given toboth the antisense compound and the antidote. For example, certainantisense compounds are RNA-like (certain such compounds may rely onRISC and/or other RNases for their activity). In certain embodiments, anantidote for such a compound could comprise 4 or more contiguousDNA-like monomers. In certain embodiments, the resulting RNA/DNA duplexcould activate RNase H, resulting in cleavage of the RNA-like antisensecompound. In certain embodiments, antidote activity does not depend onenzymatic activity. In certain such embodiments, compounds designedwithout regard for enzymatic compatibility may incorporate modificationsto improve other attributes. For example, certain motifs yieldoligomeric compounds with high affinity for a target nucleic acid, butthat are unable to elicit enzymatic cleavage of that target. Such motifsmay be useful for antidote compounds in embodiments where cleavage ofthe antisense compound is not necessary.

In certain embodiments, an antisense compound and corresponding antidotecompound are the same length. In certain embodiments, an antisensecompound and corresponding antidote compound are different lengths.

Non-limiting examples of antisense/antidote pairs is provided in thefollowing table:

Antisense Compound Antidote Compound Length Motif Length Motif 20 5-10-5MOE gapmer 20 5-10-5 MOE gapmer 20 5-10-5 MOE gapmer 20 Uniform MOE 205-10-5 MOE gapmer 20 Uniform 2'-F 20 5-10-5 MOE gapmer 18 Uniform BNA 205-10-5 MOE gapmer 20 5-10-5 LNA gapmer 16 3-10-3 MOE gapmer 16 3-10-3MOE gapmer 16 3-10-3 MOE gapmer 14 2-10-2 LNA gapmer 18 4-10-4 LNAgapmer 18 4-10-4 LNA gapmer 20 Uniform 2′-F 20 Uniform 2′-F 18 2-10-4-1LNA-DNA- 20 Uniform LNA LNA-DNA 14 2-10-2 BNA-RNA-BNA 14 Uniform BNAThe above listed pairs of antisense and antidote compounds are onlyexemplary. One of skill in the art can select any length and motif forthe sense and independently select any length and motif for the antidotecompound. The antisense and antidote compounds may, likewise comprisemodified internucleoside linkages in any combination.

Because the antidote compound is complementary to the antisensecompound, it is at least partially identical to the antisense targetnucleic acid (i.e., it is a sense strand). In certain embodiments,treatment with an antisense compound followed by an antidote compoundcould result in formation of a double-stranded duplex with antisenseactivity. For example, such a duplex could be an siRNA and activate theRISC pathway. In embodiments where a decrease of antisense activity issought, such duplexes should be avoided. Thus, in certain embodiments,where the antisense compound comprises RNA-like nucleosides suitable forloading into RISC, the antidote compound should avoid modifications thatwill allow or facilitate such loading of the antisense compound intoRISC.

In certain embodiments, an antisense compound and an antidote compoundare administered to a patient. In certain such embodiments,pharmaceutical compositions comprising an antisense compound and thosecomprising an antidote compound comprise the same formulation. Incertain embodiments, pharmaceutical compositions comprising an antisensecompound and those comprising an antidote compound comprise differentformulations. In certain embodiments an antisense compound and anantidote compound are administered by the same route. In certainembodiments an antisense compound and an antidote compound areadministered by different routes. For example, in certain embodiments,an antisense compound is administered orally and an antidote compound isadministered by injection. In certain embodiments, the dosages of theantisense compound and the antidote compound are the same. In certainembodiments, the dosages of the antisense compound and the antidotecompound are different.

In certain embodiments, the toxicity profiles of the antisense compoundand the antidote compound are similar. In certain embodiments, suchtoxicity profiles are different. For example, in certain embodiments, anantisense compound may be intended for chronic administration and theantidote compound is only intended for acute use as needed. In suchembodiments, the tolerance for toxic side-effects of the antidotecompound may be higher. Accordingly, modifications and motifs that maybe too toxic for use in an antisense compound may be acceptable in anantidote compound. For example, in certain embodiments, oligomericcompounds comprising one or more LNA nucleoside have been shown to havehigh affinity for a target nucleic acid, but in certain embodiments havebeen shown to cause toxicity at relatively low concentrations. Forcertain antisense compounds, where chronic administration is intended,certain such compounds comprising LNA may not be suitable. However, inembodiments where an antidote compound is not intended for chronicadministration, but rather for acute administration when antisenseactivity is problematic, such LNA modifications in an antidote compoundmay be acceptable. The increased affinity of LNA may improve theantidote effect and since the antidote is only administered for a shortperiod of time, and possibly when the patient is in distress, theincreased toxicity of LNA may be justified. Other high affinity, butpotentially toxic modifications are known.

In certain embodiments, an antisense activity is counteracted by anon-oligomeric antidote. For example, in certain embodiments, when thetarget nucleic acid is a target mRNA encoding a protein it is desirableto reduce the antisense activity and to increase in the amount of thetarget protein (e.g., target protein amount has gone too low, orcircumstances have changed resulting in the desire to restore targetprotein amount). In such embodiments, one may simply administer thetarget protein itself. Such administration will immediately reverse theantisense activity of target protein reduction. However, it may also bedesirable to administer an oligomeric antidote compound according to thepresent invention. For example, the target protein may have a shorthalf-life in the animal. Accordingly, to maintain the restored targetprotein concentration would require repeated administration of targetprotein until the antisense compound has cleared and normal proteinexpression is restored. In certain such embodiments, it is stilldesirable to administer an antidote compound to shorten the duration ofthe antisense activity. In certain embodiments an antidote compound isco-administered with a non-oligomeric antidote. In certain suchembodiments, the non-oligomeric antidote is a target protein. In certainembodiments, the non-oligomeric antidote compound is a protein havingsimilar physiological effect as a target protein or that stimulatesexpression of the target protein.

Research Tools

In certain instances, antisense compounds have been used as researchtools. For example, researchers investigating the function of aparticular gene product may design antisense compounds to reduce theamount of that gene product present in a cell or an animal and observephenotypic changes in the cell or animal. In certain embodiments, thepresent invention provides methods for reducing the amount of a geneproduct in a cell or animal through antisense and then reducing thatantisense activity, thereby restoring the inhibited gene product. Incertain embodiments, investigators may use such techniques tocharacterize proteins or untranslated nucleic acids. In certainembodiments, investigators may vary the amount of time between antisenseand antidote administration. In certain embodiments, such experimentsare used to investigate kinetics and/or turnover of gene products and/orcertain cellular functions.

Kits

In certain embodiments, the present invention provides kits comprisingone or more antisense compound and one or more corresponding antidotecompound. In certain embodiments, such kits are intended for therapeuticapplication. In certain embodiments, such kits are intended for researchuse.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

The nucleoside sequences set forth in the sequence listing and Examples,are independent of any modification to a sugar moiety, a monomericlinkage, or a nucleobase. As such, oligomeric compounds defined by a SEQID NO may comprise, independently, one or more modifications to a sugarmoiety, an internucleoside linkage, or a nucleobase. Oligomericcompounds described by Isis Number (Isis NO.) indicate a combination ofnucleobase sequence and one or more modifications to a sugar moiety, aninternucleoside linkage, or a nucleobase, as indicated.

EXAMPLES Example 1 Fas Antisense and Antidote Oligonucleotides

An antisense compound complementary to murine Fas and antidote compoundsto that antisense compound were synthesized using an Applied Biosystems380B automated DNA synthesizer (Applied Biosystems, Foster City,Calif.). Compound details are provided in Table 1, below.

TABLE 1 Fas antisense and antidotes SEQ ISIS # Chemistry Motif SequenceDescription ID 22023 5-10-5 MOE gap TCCAGCACTTTCTTTTCCGG Fas Antisense 1401770 5-10-5 MOE CCGGAAAAGAAAGTGCTGGA Antidote to 22023 2 401769Uniform MOE CCGGAAAAGAAAGTGCTGGA Antidote to 22026 2 29837 5-10-5 MOETCGATCTCCTTTTATGCCCG Non-sense control 3

Example 2 Fas Antisense/Antidote Treatment of Mice

Eight week old Balb/c mice (Charles River Laboratories (Willmingon,Mass.)) were injected sub-cutaneously with 35 mg/kg of ISIS 22023 in avolume of 200 ml of saline or with 200 ml of saline (control mice) everyother day for two injections.

One day after the second injection, the mice were divided into 3 groups:Group 1 received 35 mg/kg sub-cutaneous injections of ISIS 401770(5-10-5 MOE gapmer antidote to ISIS 22023) once daily for two days;Group 2 received 35 mg/kg sub-cutaneous injections of ISIS 401769(uniform MOE antidote to ISIS-22023) once daily for two days; and Group3 received sub-cutaneous injections of saline once daily for two days.All injections were in a total volume of 200 μl of sterile saline.Animals were sacrificed and livers were collected at 2, 4, 7, 10, and 14days after the first day of antidote treatment.

Example 3 Fas RNA

Total RNA was isolated from the livers using Rneasy mini kit (Quiagen).Fas RNA was assessed by quantitative real-time PCR, using standardtechniques. Results are summarized in FIG. 1.

Treatment with antisense compound ISIS 22023 resulted in reduction ofFas mRNA. Subsequent treatment with antidote compound ISIS 401770 orISIS 401769 reduced the antisense activity of ISIS 22023 (i.e.,treatment with such compounds reduced the reduction of Fas mRNA).

Example 4 Kinetics and Specificity of Fas Antidotes

Part 1. To study the kinetic of the antidote activity, eight week oldBalb/c mice were injected sub-cutaneously with 35 mg/kg of ISIS 22023 ina volume of 200 ml of saline or with 200 ml of saline (control mice)every other day for two injections.

Two days after the second injection, the mice were divided into 3groups: Group 1 received a single 70 mg/kg sub-cutaneous injection ofISIS 401770 (5-10-5 MOE gapmer antidote to ISIS 22023); Group 2 receiveda single 70 mg/kg sub-cutaneous injection of ISIS 401769 (uniform MOEantidote to ISIS-22023); and Group 3 received a sub-cutaneous injectionof saline. All injections were in a total volume of 200 μl of sterilesaline. Animals were sacrificed and livers were collected at 6 hours, 12hours and 1, 2, 6, and 14 days after antidote treatment.

Livers were collected and total RNA was analyzed as described above(Example 3). Results are shown in FIG. 2.

To test the specificity of the antisense activity and the antidoteactivity, eight week old Balb/c mice were divided into 3 groups andinjected sub-cutaneously using a first treatment (antisense stage) and asecond treatment (antidote stage) as described above in part 1, usingthe compounds in Table 2, below:

TABLE 2 Compounds to test specificity of antisense/antidote activityGroup First (antisense) treatment Second (antidote) treatment 1 22023(Fas antisense) 29837 (non-sense control) 2 29837 (non-sense control)401769 (antidote to 22023) 3 29837 (non-sense control) 401770 (antidoteto 22023)

ISIS 29837 is a 5-10-5 MOE gapmer with the same base composition as ISIS22023, but with the sequence scrambled, resulting 8 mismatches. Thus, itis not expected to be an effective antisense compound (groups 2 and 3)nor an effective antidote compound to 22023 (group 1). As shown in FIG.3, the mismatch control oligonucleotide did not provide an antidoteeffect suggesting that the antidote effect is sequence specific.

Example 5 PTEN Antisense and Antidote Oligonucleotides

An antisense compound complementary to PTEN and antidote compounds tothat antisense compound were synthesized using an Applied Biosystems380B automated DNA synthesizer (Applied Biosystems, Foster City,Calif.). Compound details are provided in Table 3, below.

TABLE 3 Fas antisense and antidote compounds SEQ ISIS # Chemistry MotifSequence Description ID 116847 5-10-5 MOE gap CTGCTAGCCTCTGGATTTGAPTEN antisense 4 126525 Uniform MOE TCAAATCCAGAGGCTAGCAGAntidote to 116847 5 401769 Uniform MOE CCGGAAAAGAAAGTGCTGGANon-sense control 6

Example 6 PTEN Antisense/Antidote Treatment of Mice

Eight week old Balb/c mice (Charles River Laboratories (Willmingon,Mass.)) were injected sub-cutaneously with 35 mg/kg of ISIS 116847(5-10-5 MOE gapmer PTEN antisense) in a volume of 200 ml of saline orwith 200 ml of saline (control mice) every other day for two injections.

Two days after the second injection, the mice were divided into 3groups: Group 1 received a single 70 mg/kg sub-cutaneous injection ofISIS 126525 (uniform MOE antidote to ISIS 116847); Group 2 received asingle 70 mg/kg sub-cutaneous injection of ISIS 401769 (Uniform MOEantidote to Fas, used here as non-sense control); and Group 3 received asub-cutaneous injection of saline. All injections were in a total volumeof 200 μl of sterile saline. Samples were collected at 12 hours and 1,2, 3, 7, and 14 days after antidote treatment.

Samples were processed as described above in Example 3. Results areshown in FIGS. 4 and 5, below.

Example 7 Prothrombin Antisense and Antidote Oligonucleotides

An antisense compound complementary to prothrombin and antidotecompounds to that antisense compound were synthesized using an AppliedBiosystems 380B automated DNA synthesizer (Applied Biosystems, FosterCity, Calif.). Compound details are provided in Table 4, below.

TABLE 4 Prothrombin antisense and antidote compounds SEQ ISIS #Chemistry Motif Sequence Description ID 401025 5-10-5 MOE gapATTCCATAGTGTAGGCCTT Prothrombin antisense 7 405277 5-10-5 MOE gapAAGGACCTACACTATGGAAT Antidote to 401025 8 405278 Uniform MOEAAGGACCTACACTATGGAAT Antidote to 401025 8

Example 8 Prothrombin Antisense/Antidote Treatment of Mice

Eight week old Balb/c mice (Charles River Laboratories (Willmingon,Mass.)) were injected sub-cutaneously with 30 mg/kg of ISIS 401025(5-10-5 MOE gapmer prothromin antisense) in a volume of 200 ml of salineor with 200 ml of saline (antisense control) twice per week for threeweeks (total of 6 injections).

Two days after the second injection, the mice were divided into 7 groupsand treated as summarized in Table 5, below. All injections weresubcutaneous and in a total volume of 200 μl of sterile saline.

TABLE 5 Prothrombin antisense and antidote compounds Group TreatmentDescription 1 Single injection of 30 mg/kg 5-10-5 MOE gap antidote of405277 2 Single injection of 60 mg/kg 5-10-5 MOE gap antidote of 4052773 Single injection of 90 mg/kg 5-10-5 MOE gap antidote of 405277 4Single injection of 30 mg/kg Uniform MOE antidote of 405278 5 Singleinjection of 60 mg/kg Uniform MOE antidote of 405278 6 Single injectionof 90 mg/kg Uniform MOE antidote of 405278 7 Single injection of salineAntidote control

Example 9 Sample Collection

Three days after antidote (or saline control) treatment, platelet poorplasma (PPP) was collected by cardiac puncture, as follows. Mice wereanesthetized and a 27 gage needle attached to a 1 ml syringe preloadedwith 65 μl of buffered citrate (0.06 Molar sodium citrate, pH 7.4) wasinserted between the ribs and into the heart. 0.6 ml of blood wasquickly withdrawn, resulting in a final ratio of nine parts whole bloodto one part citrate buffer. Mice were euthanized. The needle was removedfrom the syringe and the blood/citrate buffer sample was emptied into aplastic tube with a cap. That sample was immediately mixed by tappingand inverting the capped tube. Within four hours of cardiac puncture,the sample was centrifuged at 2000 rcg for 15 minutes at 22° C. and thetop rough plasma was removed and placed in a new tube. That rough plasmawas centrifuged a second time, and the top layer was removed and placedin a new tube. That PPP sample was aliquoted and stored at −80° C.

Immediately after euthinization, livers were collected and total RNA wasisolated from the livers using Rneasy mini kit (Quiagen).

Example 10 Prothrombin RNA

Total RNA from the livers (Example 9) were analyzed by RT-PCR. Theforward primer for those reactions was: AAGGGAATTTGGCTGTGACAA (SEQ IDNO. 9) and the reverse primer was: ACTTGGGTCCCCCTGCCTGCCX (SEQ ID NO.10). Results are shown in FIG. 6.

Example 11 Thrombin Generation

Platelet poor plasma samples from Example 9 were diluted 1:2 with salineand thrombin generation was measured using by Thrombin Generation Assay(TGA) using a Technothrombin TGA kit (Technoclone, Vienna Austria)following manufacturers instructions. Results are shown in FIG. 7.

Example 12 Prothrombin Time (PT)/Activated Partial Thromboplastin Time(aPTT)

Mouse PPP samples were assayed for PT and aPTT using an ACL 1000coagulation analyzer (IL Instrumentation, Beckman Coulter, Fullerton,Calif.) at 37° C. The PT tests were initiated using thromboplastin (DadeThromboplastin C Plus, Dade Behring Marburg GmbH, Germany) and the aPTTtests were performed by adding ellagic acid mixture (APTT-XL, PacificHemostasis, Fisher Diagnosis, Middletown Va.) and CaCl2. Pooled valuesobtained from the mice treated with saline were used as basal PT andaPTT. PT INR was calculated according to: INR=(PT/baseline PT)ISI, whereISI is the international sensitivity index of the thromboplastin used.Relative aPTT was calculated by dividing the measured values by baselinevalues. Results are shown in FIGS. 8 and 9.

Example 13 Specificity of Prothrombin Antidote Effect

To test the sequence-specificity of the observed antidote effect, thesame antidote compounds were tested for their ability to restoreprothrombin following treatment with a non-complementary antisensecompound that also targets prothrombin. Compounds are summarized inTable 6, below.

TABLE 6 Prothrombin antisense and non-corresponding antidote compoundsChemistry SEQ ISIS # Motif Sequence Description ID 401029 5-10-5 MOEGACAATCACTTTTATTGAGA Prothrombin 11 gap antisense 405277 5-10-5 MOEAAGGACCTACACTATGGAAT Antidote to 8 gap 401025 405278 UniformAAGGACCTACACTATGGAAT Antidote to 8 MOE 401025

Mice were treated and samples were obtained and assayed as describedabove (Examples 8-12). The single sub-cutaneous injections of 90 mg/kgof antidote compounds capable of reversing the antisense activity ofantisense compound 401025 (to which they are complementary) did notreverse the antisense effect of non-complementary antisense compound401029. Results for prothrombin RNA are shown in FIG. 10. Similarresults were obtained for PT-INR.

Example 14 Toxicity Studies

Toxicity of antidotes ISIS-403277 and 403728 was assessed by testingserum from animals treated with those compounds for known markers oftoxicity. The toxicity profile of those compounds was similar to thosepreviously observed for similarly modified oligonucleotides.

Example 15 In Vivo Sense-Oligonucleotide-Antidote for AntisenseInhibition of Murine Factor XI in BALB/c Mice Oligonucleotides

Chemistry SEQ ISIS # Motif Sequence Description ID 404071 5-10-5 MOETGGTAATCCACTTTCAGAGG Antisense targeting 12 gap all PS Factor XI 4040575-10-5 MOE TCCTGGCATTCTCGAGCATT Antisense targeting 13 gap all PSFactor XI 418026 5-10-5 MOE CCTCTGAAAGTGGATTACCA Antidote to 14gap all PS 404071

Treatment

The effects of antisense compounds directed to Factor XI and an antidotewere tested in BALB/c mice. In a first cohort, ISIS 404071 (antisensecompound targeted to Factor XI) was administered subcutaneously toBALB/c mice twice a week for three weeks at a dose of 40 mg/kg.Forty-eight hours after the final treatment of ISIS 404071, a singleinjection of PBS was administered subcutaneously.

In a second cohort, ISIS 404071 (antisense compound targeted to FactorXI) was administered subcutaneously to BALB/c mice twice a week forthree weeks at a dose of 40 mg/kg. Forty-eight hours after the finaltreatment of ISIS 404071, a single injection of 90 mg/kg of ISIS 418026(Antidote complementary to ISIS 404071) was administered.

In a third cohort, ISIS 404057 (antisense compound targeted to FactorXI) was administered subcutaneously to BALB/c mice twice a week forthree weeks at a dose of 40 mg/kg. Forty-eight hours after the finaltreatment of ISIS 404057, a single injection of PBS was administeredsubcutaneously.

In a fourth cohort, ISIS 404057 (antisense compound targeted to FactorXI) was administered subcutaneously to BALB/c mice twice a week forthree weeks at a dose of 40 mg/kg. Forty-eight hours after the finaltreatment of ISIS 404057, a single injection of 90 mg/kg of ISIS 418026(Antidote complementary to ISIS 404071) was administered.

Following antidote or PBS administration, a set of 4 mice from eachcohort were sacrificed at 12 hours, 1 day, 2 days, 3 days, 7 days, and14 days. Whole liver was collected for RNA analysis and PPP wascollected for aPTT analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of FactorXI. Results are presented as percent inhibition of Factor XI, relativeto PBS control. As shown in Table 7, mice treated with ISIS 404071without antidote showed progressive decrease in inhibition over the 14day observation period. However, mice treated with ISIS 404071 and itsantidote (ISIS 418026) showed an accelerated decrease in inhibition overthe 14 day observation period in comparison to mice which did notreceive antidote. Also shown in Table 7, treatment with ISIS 418026 didnot accelerate the decrease in the antisense activity of ISIS 404057.

TABLE 7 Percent inhibition of mouse Factor XI mRNA compared to PBScontrol 12 14 hours 1 day 2 days 3 days 7 days days ISIS 404071 93 90 8988 81 67 ISIS 404071 + 90 87 72 66 57 31 ISIS 418026 ISIS 404057 n.d.n.d. n.d. 95 n.d. n.d. ISIS 404057 + n.d. n.d. n.d. 97 n.d. n.d. ISIS418026 nd = no dataaPTT Assay

As shown in Table 8, mice treated with ISIS 404071 and antidote (ISIS418026) showed progressive decrease of aPTT over the 14 day observationperiod compared to mice treated with ISIS 404071 without antidote.

TABLE 8 Effect of antidote treatment on aPTT INR 12 hours 1 day 2 day 3day 7 day 14 day ISIS 404071 1.51 1.30 1.35 1.27 1.18 1.05 ISIS 404071 +1.45 1.23 1.16 1.15 1.10 0.95 ISIS 418026

Example 16 In Vivo Factor VIIa Protein-Antidote for Antisense Inhibitionof Murine Factor XI in BALB/c Mice Treatment

The effect of human Factor VIIa protein as a non-oligomeric antidote forISIS 404071 was tested in BALB/c mice. Two experimental groups of BALB/cmice were treated with 20 mg/kg of ISIS 404071, administeredsubcutaneously twice a week for 3 weeks. Two control groups of BALB/cmice were treated with PBS, administered subcutaneously twice a week for3 weeks. Thrombus formation was induced with FeCl3 in all of the miceexcept the first control group. Fifteen minutes before FeCl3 treatment,the first experimental group was treated with 5 μg/kg of human FactorVIIa protein antidote (product no. 407act, American Diagnostica Inc.).Two days after their last dose, all mice were anesthetized with 150mg/kg of ketamine mixed with 10 mg/kg of xylazine administered byintraperitoneal injection.

In mice undergoing FeCl3 treatment, thrombus formation was induced byapplying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3solution directly on the vena cava. After 3 minutes of exposure, thefilter paper was removed. Thirty minutes after the filter paperapplication, a fixed length of the vein containing the thrombus wasdissected out for platelet analysis.

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used toquantify platelets in the vena cava as a measure of thrombus formation.Results are presented as a percentage of PF-4 in antidote treated anduntreated mice, as compared to the two PBS-treated control groups. Asshown in Table 9, animals treated with human Factor VIIa proteinantidote expressed more PF-4 in comparison to animals treated with ISIS404071 alone. These data indicate that human Factor VIIa is successfulin rescuing the effect of antisense oligonucleotide inhibition.

TABLE 9 Analysis of thrombus formation by real-time PCR quantificationof PF-4 in the FeCl₃ induced venous thrombosis model Treatment PF-4 PBS− FeCl₃ 0 PBS + FeCl₃ 100 ISIS 404071 18 ISIS 404071 + hFV7a 68

1. An antidote compound comprising a modified oligonucleotide consistingof 12 to 30 linked nucleosides and having a nucleobase sequencecomplementary to an antisense compound.
 2. The antidote compound ofclaim 1, wherein the modified oligonucleotide is a single-strandedoligonucleotide.
 3. The antidote compound of claim 1, wherein theantidote compound is at least 90% complementary to the antisensecompound.
 4. The antidote compound of claim 1, wherein the antidotecompound is fully complementary to the antisense compound.
 5. Theantidote compound of claim 1, wherein at least one internucleosidelinkage is a modified internucleoside linkage.
 6. (canceled)
 7. Theantidote compound of claim 1, wherein at least one nucleoside comprisesa modified sugar.
 8. The antidote compound of claim 7, wherein at leastone modified sugar is a bicyclic sugar.
 9. The antidote compound ofclaim 7, wherein at least one modified sugar comprises a2′-O-methoxyethyl.
 10. The antidote compound of claim 1, wherein atleast one nucleoside comprises a modified nucleobase.
 11. (canceled) 12.The antidote compound of claim 1, wherein the modified oligonucleotidecomprises: a gap segment consisting of linked deoxynucleosides; a 5′wing segment consisting of linked nucleosides; a 3′ wing segmentconsisting of linked nucleosides; wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment and wherein eachnucleoside of each wing segment comprises a modified sugar. 13.(canceled)
 14. (canceled)
 15. The antidote compound of claim 1, whereineach nucleoside is modified.
 16. The antidote compound of claim 1,wherein the antisense compound is targeted to an mRNA.
 17. The antidotecompound of claim 1, wherein the antisense compound is targeted to anmRNA encoding a blood factor. 18-25. (canceled)
 26. The antidotecompound of claim 1, wherein the antisense compound is targeted to apre-mRNA.
 27. The antidote compound of claim 1, wherein the antisensecompound is targeted to a micro-RNA.
 28. The antidote compound of claim1, wherein the antisense compound is an RNase H dependent antisensecompound.
 29. The antidote compound of claim 1, wherein the antisensecompound alters splicing of a target nucleic acid.
 30. The antidotecompound of claim 1, wherein the antisense compound activates the RISCpathway.
 31. The antidote compound of claim 1, wherein the antidotecompound activates RNase H.
 32. The antidote compound of claim 1,wherein the antidote compound activates the RISC pathway. 33-59.(canceled)
 60. A kit comprising an antisense compound and an antidotecompound.
 61. A kit comprising an antidote compound and a non-oligomericantidote.
 62. The kit of claim 61 wherein the non-oligomeric antidote isa target protein.